This project focuses on HIV-1 replication in cultured cells to develop understandings of viral pathogenesis and the cellular factors that influence viral gene expression. Examples of important scientific advances that we have achieved in the 2011 to 2012 period are outlined below. RNA helicases are ubiquitous in plants and animals and function in many cellular processes. Retroviruses, such as human immunodeficiency virus (HIV-1), encode no RNA helicases in their genomes and utilize host cellular RNA helicases at various stages of their life cycle. We have continued our study of RNA helicases in HIV-1 replication. We have identified several novel RNA helicases that activate HIV-1 post-transcriptional gene expression and an RNA helicase that represses HIV-1 replication through the interferon pathway. HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q(35)VCF(38), which resembles the PP1-binding RVxF motif present on PP1-binding regulatory subunits. We have found that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. We have identified small molecule compounds that target the RVxF-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. Our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. We have constructed several HIV-1 molecular clones, each containing a discrete cellular miRNA positioned in Nef. These retroviral genomes express the inserted miRNA and are generally replication competent in T-cells. The inserted intragenomic miRNA was observed to elicit two different consequences for HIV-1 replication. First, the expression of miRNAs with predicted target sequences in the HIV-1 genome was found to reduce viral replication. Second, in one case, where an inserted miRNA was unusually well-processed by Drosha, this processing event inhibited viral replication. Our work is the first study to examine in detail the replication competence of HIV-1 genomes that express cis-embedded miRNAs. The results indicate that a replication competent retroviral genome is not precluded from encoding and expressing a viral miRNA.